Molecular Cell, Volume 57

Supplemental Information Top3-Rmi1 Dissolve Rad51-Mediated D Loops by a Topoisomerase-Based Mechanism Clare L. Fasching, Petr Cejka, Stephen C. Kowalczykowski, and Wolf-Dietrich Heyer

  Figure     1)   Figure Figure  S1   S1 (associated   with

A

B

 

 

RPA

95-­mer + Rad51

 

 

 

Fasching  et  al.

C

RPA Rad54 wt hd wt hd

-­

 

Rad54  +  dsDNA + Sgs1  (wt  or  hd)

+ Sgs1  (wt  or  hd)

Order  of  addition                                        Sgs1

 

30

%  D-­loops

25

D-­loop

20 15 10 5 0

95-­mer

Buffer

Sgs1 Sgs1hd

Topoisomers D-­loop

STR

TR

Sgs1

Sgs1hd

Buffer

DTR

STR

TR

Sgs1

Sgs1hd

Buffer

D

Sgs1

Sgs1hd

added   with  Rad54

DTR

added   with  RPA

TR:  Top3-­Rmi1

[

STR:  Sgs1-­Top3-­Rmi1 DTR:  Sgs1hd-­Top3-­Rmi1

95-­mer

5’  heterology

3’  heterology

E Normalized  D-­loops

1.25 1.00

3'-­het 5'-­het

0.75 0.50 0.25 0.00

Buffer

Sgs1 Sgs1hd

TR

STR

DTR

Figure S1. Sgs1 does not disrupt Rad51-mediated D-loops regardless of reaction order, and Top3-Rmi1 but not Sgs1 efficiently disrupts D-loops containing 5’ or 3’ heterology. A, Sgs1 does not disrupt Rad51-mediated D-loops regardless of reaction order. Reaction scheme with order of additions of Sgs1. B, Reactions and analysis as in Figure 1 D-F with 2 nM Sgs1, 2 nM Sgs1hd (Sgs1-K706A), or reaction buffer. C, Quantitation of D-loops. Shown are means ±  

standard deviations of three independent experiments. D, Top3-Rmi1 but not Sgs1 efficiently disrupts D-loops containing 5’ or 3’ heterologies. Reactions and analysis as in Figure 1 D-F with 2 nM Sgs1, 2 nM Sgs1hd (Sgs1-K706A), 2 nM Top3-Rmi1 (TR), 2 nM Sgs1-Top3-Rmi1 (STR), 2 nM Sgs1hd-Top3-Rmi1 (DTR), or reaction buffer with a 95-mer with either 25 nt heterology at the 3’ end (3’-het 120-mer) or 5’-end (5’-het 120-mer). E, Quantitation of D-loops. Shown are normalized means ± standard deviations of three independent experiments. Maximal D-loop levels for the 5’-het 120-mer are buffer: 27.5%, Sgs1: 27%, Sgs1hd: 27%, TR 16%, STR 21%, and DTR: 16%. Maximal D-loop levels for the 3’-het 120-mer substrate are buffer: 17%, Sgs1: 17%, Sgs1hd: 16%, TR 10%, STR 11%, and DTR: 9%.

 

 

 

 

 

 

 

 

 

 

 

 

Fasching  et  al.

SP  FT

A A

Fractions 25  28  31  34  37  40  43  46  49

KDa

KDa

150

150

75

Top3cd

50

B

Pool  37  -­  46

 

Pool  28  -­  36

Figure  S2  

Figure S2 (associated with Figure 2)

75

Top3cd

50

95mer 10  min + Rad51

C Topo-­ isomers D-­loop

 ScRPA Rad54   2  min Top3 HsRPA 10  min +   or 10  min SSB dsDNA Top3-­Rmi1 -­  RPA

ScRPA

HsRPA

 SSB

 -­  RPA

-­          T      TR

-­          T      TR

-­          T      TR

-­          T      TR

[

95-­mer

D Normalized  D-­loops

1.00 0.75 ScRPA

0.50

HsRPA

0.25

SSB no  RPA/SSB

0.00

Buffer

Top3 Top3-­Rmi1

Figure S2. Purification of Top3cd (Top3-Y356F) and effect of different ssDNA binding proteins. A, Top3-Y356F samples from fractions 25 – 46 collected from the SP-sepharose column separated on a 4 – 20% gradient gel (left). Top3-Y356F samples from the SP sepharose column flow through (FT), the high concentration pool (fractions 28-36) and the low concentration pool (fractions 37-46) (right). B, Reaction scheme and proteins. C, D-loop

 

dissolution by Top3 (T) and Top3-Rmi1 (TR) in the presence of yeast RPA (ScRPA; 100 nM), human RPA (HsRPA; 100 nM), E. coli SSB (SSB; 125 nM), or no ssDNA binding protein. D, Quantitation of D-loops. Shown are normalized means ± standard deviations of three independent experiments. The absolute values corresponding to maximal D-loop levels are ScRPA: 23%, HsRPA: 12%, SSB: 17%, no ssDNA binding protein: 8 %. .

 

 

 

 

Fasching  et  al.

DTR

DT STR

Topoisomers

Top3 TR

Sgs1hd

Buffer Sgs1

[

DTR

DT STR

ST

Top3 TR

Sgs1hd

Buffer Sgs1

* +

B

 

ST

Buffer

 

EcTop1

 

TR

 

EcTop1

Buffer

A

TR

  Figure     3)   Figure Figure  S3   S3 (associated   with

* +

Figure S3. S. cerevisiae Top3 does not relax supercoiled substrate under D-loop reaction conditions. A, Direct comparison of topological activity of Top3-Rmi1 (TR, 500 nM) and E. coli Top1 500 nM). All reactions were separated on 1.0% agarose gels and stained afterwards with ethidium bromide (left: positive image; right: negative image). * = relaxed plasmid DNA, + = linear plasmid DNA. B, Topological activity of Sgs1 (50 nM), Sgs1hd (Sgs1-K706A, 50 nM), Top3 (50 nM), Top3-Rmi1 (TR, 50 nM), Sgs1-Top3 (ST, 50 nM) and Sgs1hd-Top3 (DT, 50 nM), Sgs1-Top3-Rmi1 (STR, 50 nM) and Sgs1hd-Top3-Rmi1 (DTR, 50 nM) on supercoiled DNA (20 nM molecules). * = relaxed plasmid DNA, + = linear plasmid DNA.

 

Figure  S4  

 

 

 

 

 

 

 

 

 

 

 

 

 

Fasching  et  al.

Figure S4 (associated with Figure 5)

A

Human  RAD51,  RAD54,  RPA  D-­loops TOPOIII_-­RMI1-­RMI2

Topo-­ isomers D-­loop

Top3-­Rmi1

-­

-­ [

95-­mer

B

Yeast  Rad51,  Rad54,  RPA  D-­loops TOPOIII_-­RMI1-­RMI2

Topo-­ isomers D-­loop

-­

-­

Top3-­Rmi1

[

95-­mer

C

Protein-­free  D-­loops TOPOIII_-­RMI1-­RMI2

-­ Topo-­ isomers D-­loop

Top3-­Rmi1

-­

[

95-­mer

Figure S4. Representative gels for Figure 5. A. Human reconstituted D-loop system. B. Yeast reconstituted D-loop system. C. Protein-free D-loops.

 

Figure  6    

 

 

 

 

 

 

 

 

 

 

 

Fasching  et  al.

Figure S5 (associated with Figure 5)

EcTop1

Buffer

EcTop1

TRR

Buffer

+

TRR

37o    C

30o    C

+

[

Topoisomers

Figure S5. Human TOPOIIIalpha-RMI1-RMI2 does not relax supercoiled substrate under D-loop reaction conditions. Direct comparison of topological activity of TOPOIIIα-RMI1-RMI2 (TRR, 50 nM) and E. coli Top1 (500 nM) under D-loop conditions used for the yeast (300C) and human (370C) system. All reactions were separated on 1.0% agarose gels and stained afterwards with ethidium bromide. + = linear plasmid DNA.

 

Table S1: Oligonucleotides used in this study.

olWDH

Sequence 5’-> 3’

Text name

566

ATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAA

95-mer

GATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTG AGAATAGTG 1613

GACCTGATAAAGCTGTATCCGAGCATTTGTGAATTCTCCGAGT

het 95-mer

CAGCTTCTTACTCCCAAGAAGTTCGTTGGATTCGTATTCCGAA TGTTAAGAC 1614

ATTACTGTCCGTGCACGTTATTCTAATGGCAGCACTGCATAAT

5’-het 120-mer

TCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTG GTGAGTACTCAACCAAGTCATTCTGAGAATAGTG 1615

ATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAA

3’-het 120-mer

GATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTG AGAATAGTGATTACTGTCCGTGCACGTTATTC TA 1616

TAGAATAACGTGCACGGACAGTAAT

25-mer

1617

CTGTTCTTGGAAACGATATGAAC

Top3-Y356F-1

1618

GTTCATATCGTTTCCAAGAACAG

Top3-Y356F-2

 

Experimental Procedures DNA substrates. The plasmid DNA was purified using TritonX-100/SDS lysis and density gradient centrifugation in CsCl/Ethidium bromide as described (Sambrook et al., 1989). Proteins. Rad51 (Van Komen et al., 2006), Rad54 (Kiianitsa et al., 2002), RPA (Binz et al., 2006), RecA (Morimatsu et al., 1995), Sgs1 and Sgs1hd (Cejka and Kowalczykowski, 2010), Sgs1-Top3-Rmi, Sgs1hd-Top3-Rmi1 Top3-Rmi1, Top3 (Cejka et al., 2010; Cejka et al., 2012) were purified as described. The TOP3 wild type gene was mutagenized using primers olWDH1617 and olWDh1618 to generate the top3-Y356F catalytic mutant as described (Oakley et al., 2002). The DNA sequence of the entire Top3-Y356F open reading frame was confirmed by DNA sequencing. Top3-Y356K was purified as previously described for wild type (Cejka et al., 2012). Briefly, 8 g of Sf9 insect cells were collected from a 1.6 L culture transfected with a recombinant virus expressing Top3-Y356. All further steps were performed at 4oC or on ice. The pellet was incubated in 24 ml of lysis buffer (50 mM Tris pH7.5, 1 mM DTT, 1 mM EDTA) containing protease inhibitors (Sigmafast). 16 ml of glycerol and 3.12 ml 5 M NaCl was added followed by centrifugation of the lysates at 10,000 rpm in a J2-21 Beckman centrifuge. The lysate was incubated with 10 mL Glutathion S-transferase Sepharose beads (GE Healthcare) overnight. The beads were poured into a column, washed in a high salt buffer (50 mM Tris pH7.5, 5 mM β-mercaptoethanol, 1 M NaCl, 1 mM EDTA, 10 % glycerol, 1 mM PMSF, 10 µg/ml Leupeptin) then a low salt buffer (50 mM Tris pH8, 5 mM β-mercaptoethanol, 150 mM NaCl, 1 mM EDTA, 10 % glycerol, 1 mM PMSF, 10 µg/ml Leupeptin). Precision protease was loaded onto the column and incubated for 2h prior to elution with the low salt buffer. The eluate was diluted to lower the pH to 6.8 and 25 mM NaCl and loaded onto an SP-sepharose column. The column was washed with SP buffer (50 mM Tris pH6.8, 1 mM DTT, 50 mM NaCl, 10 % glycerol, 1 mM PMSF, 10 µg/ml Leupeptin) and loaded onto an FPLC and fractions were collected using an NaCl gradient from 50 to 500 mM. The fractions containing Top3-Y356F eluted at the expected salt concentration ~250 mM NaCl, which corresponds to the elution of wild type Top3 (Cejka et al., 2012). The purification yielded ~500 µg protein at 1.5 µM, which was aliquoted, snap frozen in liquid nitrogen and stored at -80oC. The purity of the Top3cd protein is documented in Figure S2. The absence of relevant contaminating activities was experimentally established (Zhang and Heyer, 2011). Purified human TOPOIIIα-RMI1-RMI2 complex was kindly provided by Dr. Ian Hickson and Dr. Kata Sarlós (University of Copenhagen, Denmark), and the purification procedure will be published elsewhere. D-loop assays. S.cerevisiae proteins: D-loop reactions were performed as previously described (Li et al., 2009). Briefly, the reaction was performed at 30oC in D-loop reaction buffer (30 mM Tris-Acetate pH7.5, 1 mM DTT, 50 µg/mL BSA, 5 mM Mg(OAc)2, 4 mM ATP, 100 mM NaOAc, 10 mM phosphocreatine and 10 U/ml creatine kinase). The end labeled 95-mer (20 nM molecules) was incubated with 0.67 µM Rad51 for 10 min to allow formation of the Rad51 filament. 100 nM RPA was added for an additional 10 min and the reaction was started by adding 112 nM Rad54 and plasmid pBSder (20 nM molecules). The D-loops formed for 2 min before addition of Sgs1, Top3, or complexes containing these proteins for an additional 10 min. The reactions were stopped and deproteinized by addition of Stop Buffer (final concentration 0.143 % SDS, 35.7 mM EDTA, 1.7 mg/mL Proteinase K) and incubation for 2 h at 37oC. The reaction products were separated in a 0.8% agarose gel at 6 V/cm for 150 min. The gel was then dried and visualized on a phosphoimager. For the time course assays, samples were collected at each indicated time point and placed into Stop Buffer. RecA: The RecA D-loops were made as previously described (McIlwraith et al., 2001). The reaction was performed at 37oC in RecA reaction buffer (50 mM Tris-Acetate pH7.5, 1 mM DTT, 100 µg/mL BSA, 2.5 mM

 

Mg(OAc)2, 2 mM ATP, 10 mM phosphocreatine and 10 U/ml creatine kinase). The end labeled 95-mer (20 nM molecules) was incubated with 0.67 µM RecA for 5 min followed by addition of a single-stranded binding protein (100 nM ScRPA, 100 nM HsRPA, 125 nM SSB) if used. The reaction continued for an additional 5 min. The D-loop reaction was initiated by addition of 12.5 mM Mg(OAc)2 and 20 nM molecules plasmid dsDNA. After one minute 0.5 nM Top3 or Top3Rmi1 was added and the reactions continued for an additional 5 min. The reactions were processed and the products were analyzed as described above. Human proteins: Briefly, the reaction was performed at 37oC in D-loop reaction buffer (35 mM Tris-Acetate pH7.5, 1 mM DTT, 100 µg/mL BSA, 2 mM Mg(OAc)2, 2 mM CaCl2, 1 mM ATP, 100 mM K+-glutamate, 10 mM phosphocreatine and 10 U/ml creatine kinase). The end labeled 95-mer (20 nM molecules) was incubated with 0.67 µM HsRAD51 for 10 min to allow formation of the Rad51 filament. 100 nM HsRPA was added for an additional 10 min and the reaction was initiated by adding plasmid dsDNA (20 nM molecules) and continued for 10 min. 2 nM Top3 or Top3-Rmi1 was added and incubated for an additional 10 min. The reactions were processed and the products were analyzed as described above. The HsRAD51-RAD54 D-loop reactions were identical to the RAD51 reactions except that 112 nM RAD54 was added after RPA and incubated for 5 min prior to addition of Top3 or Top3-Rmi1. Protein-free D-loops: RecA D-loops were produced as described above. The DNA of the D-loop reaction was purified using a G25 spin column to remove any SDS, resulting in a yield of approximately 50%. The deproteinated D-loop reactions were performed at 30oC in reaction buffer (30 mM Tris-Acetate pH7.5, 1 mM DTT, 100 µg/mL BSA, 5 mM Mg(OAc)2, 4 mM ATP, 20 mM phosphocreatine and 20 U/ml creatine kinase). 0.5 nM Sgs1, Sgs1hd, Top3 or Top3-Rmi1 was incubated with the D-loops for 10 min. The reactions were processed and the products were analyzed as described above. Supplemental references Binz, S.K., Dickson, A.M., Haring, S.J., and Wold, M.S. (2006). Functional assays for replication protein A (RPA). Methods Enzymol 409, 11-38. Cejka, P., and Kowalczykowski, S.C. (2010). The full-length Saccharomyces cerevisiae Sgs1 protein is a vigorous DNA helicase that preferentially unwinds holliday junctions. J Biol Chem 285, 8290-8301. Cejka, P., Plank, J.L., Bachrati, C.Z., Hickson, I.D., and Kowalczykowski, S.C. (2010). Rmi1 stimulates decatenation of double Holliday junctions during dissolution by Sgs1-Top3. Nature Structl Mol Biol 17, 1377-1382. Cejka, P., Plank, J.L., Dombrowski, C.C., and Kowalczykowski, S.C. (2012). Decatenation of DNA by the S. cerevisiae Sgs1-Top3-Rmi1 and RPA Complex: A Mechanism for Disentangling Chromosomes. Mol Cell 47, 886-896. Kiianitsa, K., Solinger, J.A., and Heyer, W.D. (2002). Rad54 protein exerts diverse modes of ATPase activity on duplex DNA partially and fully covered with Rad51 protein. J Biol Chem 277, 46205-46215. Li, X., Stith, C.M., Burgers, P.M., and Heyer, W.-D. (2009). PCNA is required for initiating recombination-associated DNA synthesis by DNA polymerase δ. Mol Cell 36, 704-713. McIlwraith, M.J., Hall, D.R., Stasiak, A.Z., Stasiak, A., Wigley, D.B., and West, S.C. (2001). RadA protein from Archaeoglobus fulgidus forms rings, nucleoprotein filaments and catalyses homologous recombination. Nucleic Acids Res 29, 4509-4517. Morimatsu, K., Horii, T., and Takahashi, M. (1995). Interaction of Tyr103 and Tyr264 of the RecA protein with DNA and nucleotide cofactors. Fluorescence study of engineered proteins. Eur J Biochem 228, 779-785. Oakley, T.J., Goodwin, A., Chakraverty, R.K., and Hickson, I.D. (2002). Inactivation of homologous recombination suppresses defects in topoisomerase III-deficient mutants. DNA Repair 1, 463-482.

 

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, Cold Spring Harbor LaboratoryPress). Van Komen, S., Macris, M., Sehorn, M.G., and Sung, P. (2006). Purification and assays of Saccharomyces cerevisiae homologous recombination proteins. Methods Enzymol 408, 445-463. Zhang, X.P., and Heyer, W.D. (2011). Quality control of purified proteins involved in homologous recombination. Methods Mol Biol 745, 329-343.